[Demethylation and transcription of p16 gene in malignant lymphoma cell line CA46 induced by EGCG].
The study was purposed to investigate the possible mechanism of epigallocatechin-3-gallate (EGCG) induced p16 gene demethylation and transcription regulation in the malignant lymphoma cell line-CA46. The induced growth inhibition of CA46 cells was assayed by growth curve and MTT; the DNA content of CA46 cells was analyzed by flow cytometry after being exposed to EGCG; the methylation status of the p16 gene in CA46 cell line before and after treatment with EGCG was detected by the nested-methylation specific PCR and DNA sequencing; the mRNA of p16 and DNA methyltransferases (DNMT3A and DNMT3B) gene were determined by RT-PCR. The results showed that in comparison with the control, all the 3 different concentration of EGCG were able to inhibit the growth of malignancy cell lines and increase the cell number in G(0)/G(1) phase. After treatment with EGCG for 48 hours, the methylation level was apparently attenuated in a concentration-dependent manner. Expression of p16 gene in untreated group was mild while in the treated groups it had been greatly strengthened, as compared with untreated group, the gray scale ratio of p16 to beta-actin 1 treated with EGCG (6, 12, 24) microg/ml was increased from (0.05 +/- 0. 01) to (0.19 +/- 0.03), (0.39 +/- 0.10), (0.85 +/- 0.09) respectively, exhibiting a significant difference (p < 0.05); as compared with the untreated group, after treatment with EGCG for 48 hours, the expressions of DNMT3A and DNMT3B were obviously down-regulated. It is concluded that EGCG can activate and up-regulate the expression of p16 gene mRNA which inhibits the proliferation of CA46 cell through inducing the G(0)/G(1) arrest by demethylation and/or by inhibiting DNMT3A and DNMT3B gene.
Yu
Ai-Fang
AF
Department of Hematology, Union Hospital, Fujian Medical University, Fuzhou 350001, Fujian Province, China.
Shen
Jian-Zhen
JZ
Chen
Zhi-Zhe
ZZ
Fan
Li-Ping
LP
Lin
Fu-An
FA
D002392:Catechin / Q000031:analogs & derivatives* / Q000494:pharmacology; D045744:Cell Line, Tumor; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000378:metabolism; D019175:DNA Methylation; D000090205:DNA Methyltransferase 3A; D015972:Gene Expression Regulation, Neoplastic; D019942:Genes, p16; D006801:Humans; D008223:Lymphoma / Q000235:genetics*; D014158:Transcription, Genetic; D000097584:DNA Methyltransferase 3B
D002392:Catechin; D004248:DNA (Cytosine-5-)-Methyltransferases; D000090205:DNA Methyltransferase 3A; D000097584:DNA Methyltransferase 3B; C000717967:DNMT3A protein, human; C045651:epigallocatechin gallate